Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH)

Abstract

We describe an integrated microfluidic device (μFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The μFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of μFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The μFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome.

Figure 1

Schematic of the microchip design for fluorescence in situ hybridization (FISH) and flow cytometry (µFlowFISH). (A) The mask design of the µFlowFISH chip. (B) An image of the FISH chamber formed by two photopolymerized membrane in the channel. (C) The cross-channel structure for electrokinetically focusing microbial cells into a single stream line along the center of the vertical channel for flow cytometry. The enlarged image of the channel cross shows Escherichia coli being focused in the center of the channels.

Figure 2

Schematic procedure for on-chip FISH. (A) Cell loading. Microbial cells loaded into the cell reservoir are mobilized into the FISH chamber under an electric field of 20 V/cm. The direction of the electric field is switched every 30 seconds, alternating cell loading between the two membranes. A total 10 cycles are performed. (B) Probe loading. The probes in the probe reservoir are loaded into the FISH chamber following the same method as that of cell loading, except that probes are not loaded towards the washing membrane. Twenty loading cycles are conducted, during which probes are concentrated against the loading membrane. (C) Incubation. The loaded cells and probes are incubated together near the loading membrane. After every 290 s of incubation, the mixture is pushed against the loading membrane for 10 s. (D) Washing. The cells are moved to the washing membrane in this step. Excess probes are washed through the membrane under an electric field, or diffused out of the chamber via channel inlet/outlet. The total analysis time of the on-chip FISH is about 2.5 hours.

Figure 3

µFlowFISH analysis of Escherichia coli. (A) Fluorescence image of E. coli hybridized with Alexa488-labeled Eco681 probes in the FISH chamber. Two vertical lines indicate the boundaries of the chamber. (B) Very weak fluorescence signals in the negative control using Cy3-labeleed NON338. (C) On-chip flow cytometry results of E. coli. The black line is the positive control experiment using Eco681 probes, showing about 95% of the cells were successfully labeled with probes. The red dashed line indicates that only 0.4% of the cells have strong fluorescence signals when incubated with the negative control NON338 probes. (D) Typical raw data traces obtained from the flow cytometry detection system. Unlabeled cells, as indicated in the dashed rectangles, produce only scattering peaks (bottom trace) without corresponding fluorescence peaks (top trace), while labeled E. coli cells have aligned peaks in both channels.

Figure 4

µFlowFISH analysis of cultured Hanford isolates RCH1 and RCH2. (A) On-chip FISH of cultured RCH2 using Alexa488-labeled PSM G probe. (B) On-chip flow cytometry result showing 97% of RCH2 cells were hybridized with PSM G probes and only 1.9% of cells emitted fluorescence with Alexa488-NON338 probes. (C) FISH staining of cultured RCH1 with Alexa488-labeled DSV1292 probes. (D) Flow cytometry results of the stained RCH1 cells with Alexa488-DSV1292 and Alexa488-NON338. 74% and 0.1% of the cell population are stained, respectively.

Figure 5

Analysis of a mixture of RCH1 and RCH2 at a ratio of 1:1 using µFlowFISH. (A) The FISH staining of the mixture with Alexa488-EUB338. (B) Negative control of the same mixture sample with Cy3-NON338 probes. (C) RCH2 in the mixture labeled with probe Alexa488-PSM G. (D) RCH1 in the mixture hybridized with probe Cy3-DSV1292. (E) Flow cytometry results of the mixture sample analysis where only Alexa488-PSM G probe was used to detect RCH2 cells revealing that 45 ± 10% of the cell population were stained with PSM G probes. (F) Representative flow cytometry traces showing labeled cells have both the scattering and the fluorescence peaks while unlabeled only have the scattering signals.

Figure 6

Analysis of Hanford groundwater samples using µFlowFISH. (A) On-chip FISH of the first water sample. The green spots indicate RCH2 cells were stained with Alexa488-PSM G probes. (B) The on-chip flow cytometry result of the same sample. About 12±3% of the cells are identified as Pseudomonas. In contract, the other two water samples (C and D) have no detectable Pseudomonas cells.